The PTH1R stimulates at least two effectors, adenylyl cyclase (AC) and phospholipase C, by activating Gs and members of the Gq family. Although studies have suggested links between second messenger pathways and specific actions of PTH and PTHrP in the kidney and in the skeleton, the data remain inconclusive. The major goal of this proposal is to define which actions of PTH and PTHrP are AC-independent. We will compare the biological responses elicited by PTH stimulation of two AC- defective receptor mutants, with those of the PTH1R both in vivo and in mice. Aims I and II ask whether PTH stimulates AC-independent pathways that affect osteoblast biology and osteoclastogenesis, respectively. These studies will take advantage of novel, clonal, conditionally- transformed osteoblastic and marrow stromal cell lines that have both copies of the PTH1R gene deleted. By stable transfection, the PTH1R or the AC-defective mutants will be expressed in these cells. Interactions between genetically-altered stromal cells and osteoclast progenitors will first determine whether osteoclast progenitors express the PTH1R, and then examine whether PTH affects osteoclastogenesis by stimulating AC-independent pathways. Aims III and IV complement Aims I and II in vivo, by assessing the phenotype of mice in whom both PTH1R genes has been replaced by the AC-defective mutant. We will study whether PTH or PTHrP, through AC-independent pathways, affects parameters of renal or bone function, with a special focus on establishing if specific effector pathways are linked to PTH's anabolic actions on bone. We also will test the hypothesis that PTHrP acts to block chondrocyte proliferation of growth-plate cartilage by stimulating AC-independent mechanisms. Results from these in vitro/in vivo analyses will clarify which responses to PTH are AC-independent. They also may give insight into therapeutic measures to optimize PTH's capacity to increase bone mass, and further our understanding of diseases that impair growth in children.